Abstract:
More than half of patients with late stage melanoma are found with brain metastases over the course of their treatment. Not only does this severely lessen the patient’s quality of life, but it also makes treatment more difficult as drugs have trouble crossing the blood-brain barrier (BBB). The BBB is a specialised structure that protects the brain from the potential harm of foreign bodies in the blood through high expression of junctional proteins, efflux transporters and reduced expression of apical cell adhesion molecules. The two aims of this Masters project was first, to optimise a transwell-based model of the BBB using a novel cryosectioning technique and confocal imaging, then secondly, to treat this model with melanoma cells to investigate the proteins that may be involved in the process of melanoma extravasation to the brain. Brain endothelial cells (BECs) were seeded on Alvetex scaffolds and PET transwell membranes. Immunocytochemistry was performed on the cells using a novel cryosectioning technique to provide an orthogonal view of the cells, and top-down confocal microscopy assessed the abundance and localisation of junctional proteins. New Zealand Melanoma (NZM) cells were treated to the brain endothelial cell monolayer to investigate changes in junctional protein localisation caused by this melanoma cell addition It was found that BECs expressed prominent levels of adherens junction proteins, with lower than expected expression of investigated tight junction proteins. The orthogonal view of the transwells, provided the ideal perspective to find that cells had migrated from one side of the membrane to the other, causing us to select a membrane with smaller pore sizes to avoid this migration. Melanoma cell-additions generally caused reduced junctional staining. Of interest was the finding that a subset of NZM cells may be using the junctional proteins CD144 to infiltrate the vasculature and extravasate in vivo. This data presents a BBB model capable of investigating the molecular interactions involved in melanoma metastasis. This model has found NZM cells to negatively affect endothelial barrier integrity, and NZM expression of CD144 is implicated in the process of melanoma extravasation, making it of interest for future research in this area.